CD44 Anti Human

What is CD44 and why is it significant in research?

CD44 is a single pass type I transmembrane glycoprotein widely expressed on leukocytes, erythrocytes, brain white matter, and certain epithelial cells. It functions primarily as a receptor for hyaluronic acid (HA) and mediates cell-cell interactions, adhesion, and migration. CD44 participates in lymphocyte activation, recirculation, and homing processes . Its significance in research stems from its upregulation in various carcinomas, which may correlate with metastatic potential. CD44 isoforms have emerged as important prognostic markers in head and neck, lung, colorectal, breast, and hepatocellular cancers . The protein's involvement in both normal physiological processes and pathological conditions makes it a valuable target for basic research and therapeutic development.

How do CD44 isoforms differ structurally and functionally?

CD44 exists in multiple isoforms due to alternative splicing of exons in the membrane-proximal region of the extracellular domain. The standard form (CD44s) is approximately 85-90 kDa and lacks alternative spliced products. Other isoforms range from ~90 kDa to ~220 kDa depending on splicing patterns and glycosylation levels .

Functionally, these isoforms exhibit distinct properties:

• CD44v6 contributes to cancer metastasis and functions as a co-receptor for receptor tyrosine kinases including MET and VEGFR-2

• CD44v4-7 confers metastatic phenotypes in otherwise non-metastatic cells

• CD44v3 binds to heparan sulfate-binding growth factors (FGFs, HB-EGF) and can promote tumor progression

Standard CD44 is widely expressed in normal tissues, whereas variant isoforms show more restricted expression patterns related to organ specificity and immune activation .

What criteria should guide selection of an appropriate anti-CD44 antibody clone?

Selecting an appropriate anti-CD44 antibody requires consideration of:

• Epitope recognition pattern - Different clones recognize distinct epitopes:

  • Clone 156-3C11 recognizes epitope 3 (protease-resistant) and identifies the standard form while potentially recognizing all isoforms

  • Clone F10-44-2 targets epitopes outside the variable region exons and recognizes all CD44 isoforms

  • Clone Bu52 recognizes an epitope common to all CD44 isoforms

• Experimental application - Validated applications vary by clone:

  • Western blot: Some antibodies perform optimally at specific concentrations (e.g., 0.2 μg/mL for AF3660)

  • IHC: Conditions may require optimization (e.g., 15 μg/mL antibody concentration with overnight incubation at 4°C)

  • Flow cytometry: Different clones show varying sensitivity for detecting CD44 on cancer cell lines

• Isoform specificity - Determine whether you need an antibody that:

  • Recognizes all CD44 variants (pan-CD44)

  • Targets specific variants (such as CD44v6)

  • Distinguishes between standard and variant forms

• Expected molecular weight - CD44 detection ranges from ~85 kDa (standard form) to ~153 kDa for certain variants in specific tissues .

What are optimal conditions for detecting CD44 by Western blot?

Western blot detection of CD44 requires careful optimization:

Protocol components:

• Sample preparation: Cell lysates from appropriate cell lines (HeLa, HUVEC, PC-3 show reliable expression)

• Membrane: PVDF membrane recommended

• Antibody concentration: 0.2 μg/mL (e.g., with AF3660 antibody)

• Secondary antibody: HRP-conjugated secondary matched to primary antibody species

• Detection conditions: Reducing conditions recommended for consistent results

• Buffer system: Immunoblot Buffer Group 1 has been validated

Technical considerations:

• Expected band size varies by isoform (standard CD44: ~85-90 kDa; variants: up to ~153 kDa)

• Use separation systems capable of resolving 12-230 kDa range proteins

• Some cancer cell lines (e.g., SAS and HSC-2) may show low detection sensitivity in Western blot despite confirmed expression by other methods

• Multiple bands may appear representing different isoforms or glycosylation states

How should immunohistochemistry protocols be optimized for CD44 detection?

Successful CD44 immunohistochemistry requires:

Sample preparation:

• Immersion-fixed, paraffin-embedded tissue sections yield reliable results

• Human tonsil serves as an effective positive control tissue

Staining protocol:

• Antibody concentration: ~15 μg/mL (validated with AF3660)

• Incubation: Overnight at 4°C provides optimal signal-to-noise ratio

• Detection system: HRP-DAB Cell & Tissue Staining Kit compatible with the primary antibody

• Counterstain: Hematoxylin provides appropriate contrast

• Expected pattern: Cell surface and cytoplasmic localization

Result interpretation:

• CD44 staining appears on cell surfaces and in cytoplasm

• Expression patterns may vary by tissue type and disease state

• Compare with negative controls (isotype-matched antibodies) to confirm specificity

What functional assays can evaluate the biological activity of anti-CD44 antibodies?

Several functional assays assess anti-CD44 antibody activity:

Complement-dependent cytotoxicity (CDC):

• Culture target cells (2×10^4 cells/well) in 96-well plates

• Incubate for 5 hours at 37°C with anti-CD44 antibodies and 10% rabbit complement

• Assess viability using MTS assay

3D proliferation inhibition:

• Plate cells (2,000 cells/100 μl/well) in ultra-low attachment plates

• Treat with 100 μg/ml anti-CD44 antibody

• Measure viability after 48h using CellTiter-Glo 3D reagent

• Calculate proliferation rate relative to PBS control

Adhesion inhibition:

• Anti-CD44 antibodies that neutralize hyaluronic acid binding inhibit anchorage-independent growth of carcinoma cells

• Assess cell attachment to hyaluronic acid-coated surfaces with and without antibody

In vivo models:

• Anti-CD44 antibodies exhibit significant antitumor activity in mouse xenograft models of human cancers

• Measure tumor growth, metastasis, and survival endpoints

How can researchers distinguish between CD44 isoforms experimentally?

Differentiating CD44 isoforms requires strategic experimental approaches:

Western blot analysis:

• Different isoforms appear at distinct molecular weights

• Standard form: ~85-90 kDa

• Variant isoforms: Up to ~153 kDa or higher depending on glycosylation and splicing

• Use gradient gels for optimal separation

RT-PCR analysis:

• Multiple bands represent different splice variants

• Design primers flanking variable regions

• Cancer cell lines like SAS and HSC-2 show multiple CD44v bands in RT-PCR despite low protein detection by Western blot

Flow cytometry:

• Use antibodies recognizing common regions to detect all CD44 forms

• Compare with variant-specific antibodies to determine isoform expression profiles

• Both C44Mab-5 and 5-mG2a-f antibodies showed high sensitivity for detecting CD44 on CHO/PA16-CD44v3-10, SAS, and HSC-2 cells

Immunohistochemistry:

• Compare staining patterns using pan-CD44 versus variant-specific antibodies

• Analyze co-localization with variant-specific markers

What challenges exist in targeting CD44 with therapeutic antibodies?

Therapeutic targeting of CD44 presents several challenges:

Target expression on normal tissues:

• CD44 is widely expressed on leukocytes, erythrocytes, and epithelial cells

• Potential for off-target effects and toxicity

Isoform heterogeneity:

• Multiple CD44 variants with different functions complicate targeting strategies

• Cancer cells may express multiple isoforms simultaneously

Need for cancer-specific targeting:

• Development of cancer-specific anti-CD44 antibodies (CasMabs) could reduce adverse effects

• Similar approaches have been successful with other targets like podoplanin

Multi-target requirement:

• "Targeting multiple targets, such as EGFR, HER2, PODXL, and CD44 may be needed for effective therapy to conquer oral cancers"

• Single-target approaches may have limited efficacy due to pathway redundancy

Antibody engineering considerations:

• Modifications like defucosylation (as in 5-mG2a-f) can enhance antibody-dependent cellular cytotoxicity

• Optimal effector functions may vary by cancer type and therapeutic goal

How do post-translational modifications affect CD44 antibody recognition?

Post-translational modifications significantly impact CD44 antibody recognition:

Glycosylation:

• CD44 undergoes extensive N-glycosylation and O-glycosylation

• Glycosylation patterns can mask epitopes or create neo-epitopes

• Cancer-associated glycosylation changes may alter antibody binding

• Molecular weight varies substantially depending on glycosylation status

Proteolytic processing:

• CD44 can undergo ectodomain shedding

• Antibodies targeting regions affected by proteolysis may show inconsistent binding

Other modifications:

• Sulfation affects ligand binding properties

• Phosphorylation modulates signaling functions

• These modifications can alter protein conformation and antibody accessibility

Technical implications:

• Deglycosylation enzymes may be necessary to study core protein

• Multiple antibodies recognizing different epitopes provide more comprehensive analysis

• Culture conditions can affect modification patterns, requiring standardization

What are common causes of inconsistent CD44 antibody staining?

When CD44 antibody staining yields inconsistent results, consider:

Epitope accessibility issues:

• Heavy glycosylation may mask epitopes

• Try different antigen retrieval methods (heat-induced or enzymatic)

• CD44 isoform variability affects epitope availability

Expression level variation:

• CD44 expression can be low in some cancer cell lines, detectable by flow cytometry but not by Western blot (as observed with SAS and HSC-2 cells)

• Use more sensitive detection methods for low-expressing samples

• Standardize culture conditions as expression may vary with cell density

Antibody clone specificity:

• Different clones have varying affinities and epitope recognition patterns

• Clone 156-3C11 recognizes epitope 3 (protease-resistant)

• Clone F10-44-2 recognizes epitopes outside variable regions

• Clone Bu52 recognizes common epitopes across isoforms

Fixation and processing effects:

• Some epitopes are fixation-sensitive

• Compare results across different fixation methods

• Consider native versus denatured conformation requirements

How can researchers validate anti-CD44 antibody specificity?

Robust validation approaches include:

Positive and negative controls:

• Positive controls: HeLa, HUVEC, PC-3, human tonsil

• Negative controls: CHO-K1 cells (unreactive with C44Mab-5 and 5-mG2a-f)

• Include isotype controls at equivalent concentrations

Multi-technique validation:

• Compare results across different applications (Western blot, flow cytometry, IHC)

• Flow cytometry may detect CD44 when Western blot fails to show bands

• RT-PCR confirms expression at mRNA level when protein detection is challenging

Cross-reactivity assessment:

• Test for species cross-reactivity (e.g., AF3660 shows ~25% cross-reactivity with mouse CD44)

• Evaluate binding to related proteins

Antibody-specific characteristics:

• Understand each antibody's properties:

  • 156-3C11: Mouse IgG2a against CD44, recognizes epitope 3

  • F10-44-2: Mouse IgG2a against CD44, recognizes epitopes outside variable regions

  • Bu52: Mouse IgG1 against CD44, recognizes common epitopes

What emerging approaches enhance anti-CD44 antibody applications in cancer research?

Novel approaches to enhance anti-CD44 antibody utility include:

Glycoengineered antibodies:

• Defucosylated antibodies like 5-mG2a-f enhance effector functions

• Glycomodification optimizes antibody-dependent cellular cytotoxicity

Cancer-specific antibodies:

• Development of cancer-specific anti-CD44 mAbs (CasMabs)

• Target cancer-specific epitopes or CD44 conformations

• Similar approaches with podoplanin (PDPN) show promise in oral cancer models

Combination targeting strategies:

• Multi-target approaches addressing CD44 along with EGFR, HER2, and PODXL

• Combinatorial antibody cocktails for enhanced efficacy

• Integration with existing therapeutic modalities

Application-specific optimization:

• For diagnostics: Higher sensitivity detection methods

• For therapeutics: Enhanced tumor penetration and reduced off-target effects

• For research: Improved isoform discrimination and functional studies

Technological innovations:

• Antibody fragments with improved tissue penetration

• Bispecific antibodies targeting CD44 and secondary cancer markers

• Antibody-drug conjugates for targeted therapy

Table of CD44 Antibody Clones and Applications